AS201

BACHELOR OF SCIENCE (HONS) BIOLOGY

MICROBIOLOGY BIO461
LAB REPORT 1

NAME: ABDUL HAKIM HAYY BIN ZUBER

MATRIC NUMBER: 2022868408
CLASS: AS2011B
OBJECTIVES:
1. To compare stained cells between bacterial cells and typical plant and animal cells.
2. To observe the living motile and non-motile bacteria.
3. To identify certain internal structures by staining the organism before observing them.

A. EXAMINATION OF STAINED CELLS

Materials.
1. Compound microscope
2. Staphylococcus subtilis
3. Bacillus subtilis
4. Salmonella typhimurium

Procedure
1. All the slides have been observed under the oil immersion objectives.
2. The size and shape and any visible structures of these organism has been observed.
3. Illustration or drawing of all observation has been made.

B. EXAMINATION OF LIVING BACTERIA

MATERIALS:
1. 24- hour nutrient broth cultures of
(a) Staphylococcus aureus
(b) E. coli
2. Hollow ground depression slides
3. Vaseline
4. Inoculating loops
5. Glass slides and cover slips
6. Applicator sticks
I. HANGING DROP TECHNIQUE

Procedure
1. Vaseline has been applied to the edges on one surface of the cover slip with the aid of a
wooden applicator stick and has been placed on the lab table so that the Vaseline
treated surface faces upward.
2. A drop of bacterial broth culture has been placed on the center of the cover slip using a
sterile inoculating loop.
3. A depression slide has been inverted and lowered onto the prepared cover slip. The
slide has been gently pressed down so that the Vaseline forms a seal between the slide
and the coverslip.
4. The slide has been carefully turned over, making sure that the drop does not run
sideways.
5. Focus on the drop using the x10 objectives. Change to the x40 objectives and reduce the
light intensity.
6. The type of movement has been observed:

a. Drifting of the bacteria across the field of view caused by evaporation of the
drop through an incomplete Vaseline seal.
b. Brownian motion.
c. True motility-a characteristic of flagellated bacteria. The bacteria have been
observed darting about and changing direction.
7. Findings has been observed.

II. TEMPORARRY WET MOUNT TECHNIQUE

Procedure
1. The inoculating loop has been flamed to redness.
2. The plug has been removed from the bacterial culture to be used and the mouth of the
tube has been flamed.
3. A loopful of the culture has been removed and has been placed in the center of a clean
glass slide.
4. The mouth of the tube has been flamed and the plug has been returned.
5. The inoculating loop has been sterilized.
6. A cover slip has been put over the bacterial suspension and has been pressed down
gently
 7. The preparation has been examined under low power and then under high power
objectives.

C. THE STAINING OF MICROORGANISMS

PREPARATION OF FILMS FOR STAINING

I. From broth cultures
1. An inoculating loop has been sterilized, allow it to cool and one drop of bacterial
suspension from the tube has been removed.
2. The drop has been placed in the center of a clean, grease free microscope slides and the
drop has been spread out to form a thin film towards the top end of the slide. Do not go
too near the edges of the slide.
3. The film has been allowed to dry by wafting the slide gently above a Bunsen flame.
4. The dried smear, film uppermost has been passed quickly once or twice through the
Bunsen flame.
5. The slide has been placed on a staining rack and the stains has been applied.

II. From agar cultures

1. A drop of sterile water has been placed in the center of a slide.
2. The loop has been sterilized and a minute quantity of the bacterial culture has been
removed.
3. The cells have been mixed in the drop of water and proceed as in the case of broth
cultures. (Steps 2-5)

DIRECT STAINING WITH BASIC DYES

SIMPLE STAINING TECHNIQUES

Materials
1. 24 hours broth cultures of
a. E. coli
b. Staph. Aureus
2. 24 hours nutrients agar slants of
a. Bacillus subtilis
b. Pseudomonas aeruginosa
3. Slides
4. Inoculating loop
5. Dyes solutions
a. crystal violet
b. methylene blue
c. carbon fuchsin
6. Test tubes

Procedure
1. Some saliva has been collected in the test tube provided.
2. The film has been prepared for staining.
3. The film has been flooded with approximately 5 drops of the stain and allow time for
the stains to react:
Methylene blue 30 seconds, crystal violet 10 seconds, carbon fuchsin 5 seconds.
4. The stain has been washed off with tap water, excess water has been blotted off and
the stained film high has been dried over a Bunsen flame.
5. The slide has been examined directly without the cover slip under the oil immersion
objective lens (x100).
6. Labeled diagram has been drawn to show the morphological shapes of the bacterial
cells.
7. Results has been recorded
RESULTS

A. EXAMINATION OF STAINED CELLS

1. Staphylococcus aureus
2. Bacillus subtilis
3. Salmonella typhimurium
B. EXAMINATION OF LIVING BACTERIA

I. HANGING DROP TECHNIQUE

1. Bacillus subtilis

100x magnification
2. E. coli

100x magnification
II. TEMPORARY WET MOUNT TECHNIQUE
1.Bacilus subtilis

100x magnification
2. E. coli

100x magnification
C. THE STAINING OF MICROORGANISM
1. Bacillus subtilis

4x magnification

10x magnification
 40x magnification

100x magnification
2. E. coli

100x magnification
Discussion
Every organism has their own size, shape, and visible structures. Staphylococcus aureus is
coccus shaped and they grow in clusters, pairs, and occasionally short chains. Bacillus subtilis is
a rod-shaped cell. Salmonella typhimurium is a short, rod-shaped bacteria.
There are motile and non-motile bacteria living around us such as Bacillus subtilis and E. coli.
Bacillus subtilis use hair-like extension called flagella or cilia for movement. It is capable of
swimming in liquid medium, propelling itself by means of multiple, rotating flagella, which are
displayed peritrichously around the cell. E. coli is propelled by a bundle composed of multiple
flagella. It moves with the help of helical flagella in an aquatic environment. Helical flagella are
rotated in clockwise or counterclockwise direction using reversible flagella motors situated at
the base of each flagellum.
Living bacteria is almost colorless, and do not present sufficient contrast with the water in
which they are suspended to be clearly visible. The purpose of staining is to increase the
contrast between the organism and the background so that they are more readily seen in the
compound microscope.

Conclusion
We can observe microorganism to identify the size, shape, and visible structures. We can also
know that there are motile and non-motile bacteria. By staining the bacteria, we can clearly see
the size, shape, and visible structures.
References

 Kumar, M. S., & Philominathan, P. (2010, February). The physics of flagellar motion
of E. Coli during chemotaxis. Biophysical reviews. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418374/#:~:text=coli%20moves%20w
ith%20the%20help,is%20unique%20and%20time%20reversible
 Encyclopædia Britannica, inc. (n.d.). Coccus. Encyclopædia Britannica. Retrieved from
https://www.britannica.com/science/coccus-bacterial-shape
 Libretexts. (2021, May 26). 4.1: Introduction to staining. Biology LibreTexts. Retrieved
from
https://bio.libretexts.org/Courses/North_Carolina_State_University/MB352_General_M
icrobiology_Laboratory_2021_(Lee)/04%3A_Staining_Techniques/4.01%3A_Introductio
n_to_Staining